Enterobacteria. Escherichia coli. Shigella. Enteritis & Bacterial Dysentery

1. THEME: PATHOGENIC ENTEROBACTERIACEAE. ESCHERICHIA COLI.
SHIGELLA. MORPHOLOGY AND BIOLOGICAL PROPERTIES. LABORATORY
DIAGNOSTICS OF ENTERITIS AND BACTERIAL DYSENTERY.
I. THEORETICAL QUESTIONS
1. General characteristics of family Enterobacteriaceae. Morphology, cultural
characteristics, antigen structure, resistance, and significance in human pathology.
2. Escherichia coli and Shigella spp. Morphology, cultural features, antigen structure
and serological classification.
3. Classification of E.coli according to pathogenicity. Virulent factors of E.coli.
4. Epidemiology and pathogenesis of diarrhea caused by E.coli.
5. Laboratory diagnostics of the coli-enteritis.
6. Cultural method
7. Prevention and control of the diarrhea provoked E.coli.
8. Epidemiology and pathogenesis of bacterial dysentery. Laboratory diagnostics of
the bacterial dysentery
i. Culture method
ii. Serological method
9. Specific prophylaxis and treatment of shigelosis.
The family Enterobacteriaceae includes many Gram-negative, as a rule, motile, nonspore-
forming, facultative bacilli with simple growth requirements.
The majority of Enterobacteriaceae are normal gastrointestinal flora. In the gastrointestinal tract
they exist in a symbiotic relationship with the host. They synthesize vitamin K, and spleat
bile salts and sex hormones for recirculation to the liver. They also prevent the
colonization of intestinal mucosa by primary pathogens. Colonization is inhibited by
colicin/bacteriocin synthesis and receptor competition.
The normal flora of Enterobacteriaceae, which include Escherichia, Citrobacter,
Klebsiella, Enterobacter, Serratia, and Proteus, generally lack the virulence factors of the
pathogenic species. They act as opportunistic pathogens when they invade the normal
anatomic barriers or the host is severely immunocompromised.
Opportunistic Enterobacteriaceae are the most common cause of intra-abdominal
sepsis, and the most common cause of urinary tract infections, A subset of
Enterobacteriaceae are considered primary pathogens because they contain virulence
factors capable of overcoming normal host defences. They are not part of the normal
flora. This subset includes Shigella species, Salmonella species, Yersinia species, and
some strains of Escherichia coli.
A. Morphology: The Enterobacteriaceae are Gram-negative bacilli that are motile with
peritrichous flagella or non- motile (Klebsiella and Shigella are nonmotile), 2-4 x 0.6 μm in
diameter, non-sporeforming, a few of them are capsulated (Klebsiella).
B. Cultural characteristics and biochemical activity.
1. The Enterobacteriaceae are facultative anaerobs, easily cultivated on the simple
media.
2. Members of the most genera form circular, 1-3 mm in diameter, low convex, smooth
surfaced colonies with entire edges, colourless to grayish, and semitranslucent.
3. All members of Enterobacteriaceae ferment glucose and reduce nitrates to nitrites.
4. All are oxidase negative

2. 5. Genera may differ in lactose metabolism, an important property used during
identification. Lactose fermentation may be determined on the special media which
contain lactose and indicator. Fermentation lactose is accompanied with acid
formation; therefore colonies of lactose-positive enterobacteria will be colored after
overnight incubation. Lactose-negative colonies will stay uncolored or pale..
Enterobacteriaceae can be divided into lactose fermenters and nonlactose
fermenters by their colony appearance on. The most commonly used such
selective/differential media are eosin methylene blue agar (EMB), Endo and MacConkey's
agar. If the lactose is fermented, the colony changes color.
a. Important lactose fermenters are Escherichia coli, Citrobacter, Klebsiella, Serratia, and
Enterobacter.
b. Important non-lactose fermenters include Shigella, Salmonella, Proteus, and Yersinia.
Resistance:
Enterobacteriaceae are easily destroyed by heat and by common disinfectants or germicides.
They are sensitive to drying or desiccation. In contrast, they survive best in a high moisture
environment. Respiratory care or anesthesia equipment is common source of nosocomial
infections. Contaminated ice machines or water supplies may harbor these organisms, causing
epidemics.
Antigenic structure:
1. K antigens are polysaccharide capsular antigens. They are antiphagocytic by blocking the
access of complement or antibodies to the organism. The K1 antigen of nephritogenic E. coli
strains is associated with adherence to lectins of the GU epithelium. K1 is also associated
with neonatal meningitis since it is antiphagocytic, anticomplementary, and resembles
sialic acid. As a rule, K-antigen is responsible for type specifity.
H antigens are the flagellar proteins and are present only in motile organisms. They are
typospecific antigens. H antigen may exist in one of two phases in Salmonella.
O antigen or somatic heat stable antigen is a polymer of repeating oligosaccharide units of
three or four monosaccharides. These antigens are part of the LPS found in the outer
membrane. Certain O antigens promote adherence to gastrointestinal or
genitourinary epithelium. It is a group specific antigen.
Vi antigen is revealed in some genera. It is superficial antigen, polymers of glucuronic acid,
termolabile.
Pathogenicity is due primarily to endotoxin (also called LPS; the lipopolysaccharide portion of the
cell wall) present in all Enterobacteriaceae.
Virulent factors:
1. Endotoxins. Toxicity lies in the lipid A portion. LPS may cause endotoxic shock when the
enteric bacilli enter the bloodstream (septic shock). Endotoxic shock is characterized by blood
pools forming in the microcirculation resulting in hypotension. Vital organs lack adequate
blood supply, leading to decreased tissue perfusion, acidosis, ischemia, and cellular hypoxia. DIC
may also occur to further compromise the patient.
Enterotoxins are produced by some members of the family (some f. coli, Shigella) that exert
their toxic effects on the small intestine. Enterotoxins cause secretion of fluid into the
lumen, resulting in secretory diarrhea.
Pili or fimbriae promote adhesion to tissues.
Escherichia coli.

3. E. coli are motile, lactose-positive (which helps differentiate E. coli from Shigella and Salmonella),
and will form colored colonies EMB or MacConkey's agar. The gastro-intestinal tract is a natural
reservoir for E. coli.
Morphology and cultural characteristics are common with family features. They have three types of
antigens (somatic, capsular and flagella) which are revealed with agglutination test. Serotyping of
the clinical isolates are used for identification of E.coli.
Pathogenicity is related to the presence of the capsular antigen, which inhibits phagocytasis and
is frequently found in bacteremia and meningitis. Nephropathogenicity is associated with
plamid-mediated hemolysin production. Enterotoxins (also encoded by plamids) stimulate
secretion of water and ions into the lumen of the gut, resulting in diarrhea. Heat-labile
toxin, like cholera toxin, activates adenyl cyclase by ribosylation of G proteins.
E.coli causing diarrhea is divided into five groups according to their pathogenecity and
clinical appearance of diseases.
1. Enteropathogenic E.coli (EPEC) – they cause enteritis in infants. EPEC adhere tightly to
enterocytes, leading to inflammatory reactions and epithelial degenerative changes.
Enteropathogenic Escherichia coli causes non-inflammatory diarrhea. Infants, especially in
the developing world, are very susceptible. It is more invasive than ETEC or EAggEC,
but l e s s invasive than the pathogens causing dysentery.This bacteria has no known
toxins, but several kinds of adhesins.
2. Enterotoxigenic E.coli (ETEC) – these are the strains that form a enterotoxin. ETEC cause a
diarrhoea similar to that produces by V.cholerae. Enterotoxigenic Escherichia coli (ETEC) is a
major cause of infant death in developing countries, and is the most common cause of "traveler's
diarrhea."
More than 100 serotypes of E. coli cause this non-inflammatory, secretory
diarrhea which is similar to cholera, but less severe. The organism is acquired
through the ingestion of feces-contaminated food or water. Disease is noninvasive
and occurs in the small intestine. Major virulence factors are plasmid encoded
including enterotoxins and pili-termed colonization factor antigens (CFA) that act
as adhesions
(1) Heat-labile toxin (LT) ADP ribosylates G stimulatory (Gs) protein, destroying
its GTPase activity. Without it, the alpha subunit of Gs constantly stimulates
adenylate cyclase to produce
cyclic-AMP, leading to a loss of electrolytes and water.
(2) Heat-stable (ST) toxin is a small peptide toxin that mimics the peptide
hormone guanylin. It binds to the guanylate cyclase receptor and increases the
celluar cGMP level causing mucosal cells to release fluids.
3. Enteroinvasive E.coli (EIEC) – some strains of E.coli invade the intestinal epithelial cells as
do dysentery bacilli and produce disease clinically indistinguishable from shigella
dysentery. It lacks Shiga toxin, but contains a virulence plasmid that is essentially
identical to those of Shigella. The organism is acquired through the ingestion of
contaminated food. It produces a severe, but self-limited dysentery in most adults,
but is often fatal in young children.
4. Enterohaemorrhagic E.coli (EHEC) – these strains haemorrhagic colitis and haemolytic
uraemic syndrome. It causes bloody diarrhea, similar to dysentery.
Its reservoir is livestock, especially beef cattle. The organism is acquired by
ingestion of poorly-cooked ground beef. Epidemics associated with fast-food
chains or processed food has occurred, although the organisms has also been
transmitted in milk, cider and water. One serotype, O157:H7, is the predominant

4. cause of disease. It is not significantly invasive, causing local mucosal
disease only. Virulence factors include adhesins that efface the
microvili and a bacteriophage-encoded Shiga-like
cylotoxin.
5. Enteroaggregative E.coli (EAggEC) – these strains are so named because they appear
aggregated in a “stacked brick” formation on Hep-2 cells or glass. Enteroaggregative
Escherichia coli (EAggEC) causes persistent watery diarrhea in children and traveler's
diarrhea. The organism is acquired by ingestion. Virulence factors include pili, an
enterotoxin, anda cytotoxin. The p i l i promote adherence. The enterotoxin is similar
to ST toxin
Other clinical manifestations of E. coli infection
a. E. coli is the most common causative organism in urinary tract infections (UTIs).
b. Neonatal pneumonia is usally the result of nosocomial infection due to aspiration
during the birth process. Sepsis may also occur.
c. Neonatal meningitis is also caused by exposure during birth. It is a serious infection
resulting in 40-80% mortality.
Therapy and prevention. Antibiotic therapy is based on the site, severity, and sensitivity
of the infectious organism.
a. UTI is usually treated with Bactrim (sulfa + TMP) or a fluoroquinolone.
b. Pneumonia, meningitis, and sepsis are commonly treated with a third-generation
cephalosporin such as cefotaxime and/or an aminoglycoside.
c. Diarrheal syndrome is usually treated with fluids and electrolytes only. Bismuth
subsalicylate activates and binds enterotoxins.
d. To recover intestinal microbiota after enteritis some microbial medicine should be
administered (so named eubiotics)
Laboratory diagnostics is based on culture (bacteriological) method only. Collected
specimens such as feces, vomiting, food, polluted water are cultivated on the
diagnostic lactose media. Pure culture is identified according to biochemical features
(E.coli breaks down lactose, glucose, maltose, mannitol, but does not split succhrose.
It produces H2S but not indol). Serotyping is the last step of identification.
Shigella
Structure, Classification, and Antigenic Types: Organisms of the genus Shigella belong to the
tribe Escherichia in the family Enterobacteriaceae. The genus Shigella is differentiated into four
species: S dysenteriae (serogroup A, consisting of 12 serotypes); S flexneri (serogroup B,
consisting of 6 serotypes); S. boydii (serogroup C, consisting of 18 serotypes); and S sonnei
(serogroup D, consisting of a single serotype). Shigellae are Gram-negative, nonmotile,
facultatively anaerobic, non-spore-forming rods. Shigella are differentiated from the closely
related Escherichia coli on the basis of pathogenicity, physiology (failure to ferment lactose or
decarboxylate lysine) and serology. The genus is divided into four serogroups with multiple
serotypes: A (S dysenteriae, 12 serotypes); B (S flexneri, 6 serotypes); C (S boydii, 18
serotypes); and D (S sonnei, 1 serotype). The division is based on the O-antigen structure (group-
specific and type specific fractions).
Cultivation : They scanty grow onto the differential enteric media (Endo, Ploskirev, EMB agar)
with colorless colony formation due to inability to break down lactose. Sometimes for shigella
isolation from the feces it is necessary to use enrichment media (selenite broth or agar). The
biochemical properties are used to identify species of Shigella. The S. dysenteriae splits only
glucose with acid formation, the S flexneri splits glucose, mannose and maltose as the S. boydii

5. does, but S sonnei is the most biochemically active and it splits all sugars from the short Hiss
media row (lactose and sucrose are broken down slowly after 48 hrs )
Epidemiology and pathogenesis: The source of infection is either ill person or carrier. The
bacteria are shed with feces and contaminate the water, food and soil. The healthy person is
infected by ingestion or due to poor sanitation. Shigellosis is endemic in developing countries
were sanitation is poor. In developed countries, single-source, food or water-borne outbreaks
occur sporadically, and pockets of endemic shigellosis can be found in institutions and in remote
areas with substandard sanitary facilities.
Infection is initiated by ingestion of shigellae (usually via fecal-oral contamination). An early
symptom, diarrhea (possibly elicited by enterotoxins and/or cytotoxin), may occur as the
organisms pass through the small intestine. The hallmarks of shigellosis are bacterial invasion of
the colonic epithelium and inflammatory colitis. Colitis in the rectosigmoid mucosa, with
malabsorption, results in the characteristic sign of bacillary dysentery: scanty unformed stools
tinged with blood and mucus.
Clinical Presentation: Shigellosis has two basic clinical presentations: (1) watery diarrhea
associated with vomiting and mild to moderate dehydration, and (2) dysentery characterized by a
small volume of bloody, mucoid stools, and abdominal pain (cramps and tenesmus). Shigellosis
is an acute infection with onset of symptoms usually occurring within 24-48 hours of ingestion
of the etiologic agent. The average duration of symptoms in untreated adults is 7 days, and the
organism may be cultivated from stools for 30 days or longer.
Host Defenses and Immunity: Inflammation, copious mucus secretion, and regeneration of the
damaged colonic epithelium limit the spread of colitis and promote spontaneous recovery.
Serotype-specific immunity is induced by a primary infection, suggesting a protective role of
antibody recognizing the lipopolysaccharide (LPS) somatic antigen. Other Shigella antigens
include enterotoxins, cytotoxin, and plasmid-encoded proteins that induce bacterial invasion of
the epithelium. The protective role of immune responses against these antigens is unclear.
Immunity is type-specific, weak and short-lasting.
Laboratory diagnosis: Culture method: Although clinical signs may evoke the suspicion of
shigellosis, diagnosis is dependent upon the isolation and identification of Shigella from the
feces. Positive cultures are most often obtained from blood-tinged plugs of mucus in freshly
passed stool specimens obtained during the acute phase of disease. Rectal swabs may also be
used to culture. Isolation of shigellae in the clinical laboratory typically involves an initial
streaking for isolation on differential/selective media with aerobic incubation to inhibit the
growth of the anaerobic normal flora. Commonly used primary isolation media include
MacConkey, Endo Agar, and Salmonella-Shigella (SS) Agar. These media contain bile salts to
inhibit the growth of other Gram-negative bacteria and pH indicators to differentiate lactose
fermenters (Coliforms) from non-lactose fermenters such as shigellae. Following overnight
incubation of primary isolation media at 37° C, colorless, non-lactose-fermenting colonies are
streaked and stabbed into tubed slants of Kligler's Iron Agar or Triple Sugar Iron Agar. In these
differential media, Shigella species produce an alkaline slant and an acid butt with no bubbles of
gas in the agar. This reaction gives a presumptive identification, and slide agglutination tests
with antisera for serogroup and serotype confirm the identification.
Molecular method: Sensitive and rapid methodology for identification of both EIEC and
Shigella species utilizes DNA probes that hybridize with common virulence plasmid genes or
DNA primers that amplify plasmid genes by polymerase chain reaction (PCR). Enzyme-linked
immunosorbent assay (ELISA) using antiserum or monoclonal antibody recognizing Ipa proteins

6. can also be used to screen stools for enteroinvasive pathogens. These experimental diagnostic
techniques are useful for epidemiological studies of enteroinvasive infections, but they are
probably too specialized for routine use in the clinical laboratory.
Treatment : Effective antibiotic treatment reduces the average duration of illness from
approximately 5-7 days to approximately 3 days and also reduces the period of Shigella
excretion after symptoms subside. Absorbable drugs such as ampicillin (2 g/day for 5 days) are
likely to be effective when the isolate is sensitive. Trimethoprim (8 mg/kg/day) and
sulfamethoxazole (40 mg/kg/day) will eradicate sensitive organisms quickly from the intestine,
but resistance to this agent is increasing. Ciprofloxacin (1 g/day for 3 days) is effective against
multiple drug resistant strains. Shigellosis can be correctly diagnosed in most patients on the
basis of fresh blood in the stool. Neutrophils in fecal smears is also a strongly suggestive sign.
Nonetheless, watery, mucoid diarrhea may be the only symptom of many S sonnei infections,
and any clinical diagnosis should be confirmed by cultivation of the etiologic agent from stools.
Control : Prevention of fecal-oral transmission is the most effective control strategy. Severe
dysentery is treated with ampicillin, trimethoprim-sulfamethoxazole, or, in patients over 17 years
old, a 4-fluorquinolone such as ciprofloxacin. Vaccines are not currently available, but some
promising candidates are being developed.
II.Students practical activities:
1. Prepare smear from pure culture of E.coli, stain with Gram
and microscopy. Estimate the morphology and sketch the
image. Microscopy the smears prepared from Shigella culture
2. Familiarize with diagnostic media for cultivation of E.coli.
Indicate the colonies of lactose-positive and lactose-negative
enterobacteria.
3. Detect the biochemical features of E.coli and Shigella spp.
according to growth results into the Hiss media. Note them in
protocol.
4. Write down the scheme of laboratory diagnostics of the
diarrhea caused by E.coli and bacterial dysentery caused by
Shigella spp...

7. can also be used to screen stools for enteroinvasive pathogens. These experimental diagnostic
techniques are useful for epidemiological studies of enteroinvasive infections, but they are
probably too specialized for routine use in the clinical laboratory.
Treatment : Effective antibiotic treatment reduces the average duration of illness from
approximately 5-7 days to approximately 3 days and also reduces the period of Shigella
excretion after symptoms subside. Absorbable drugs such as ampicillin (2 g/day for 5 days) are
likely to be effective when the isolate is sensitive. Trimethoprim (8 mg/kg/day) and
sulfamethoxazole (40 mg/kg/day) will eradicate sensitive organisms quickly from the intestine,
but resistance to this agent is increasing. Ciprofloxacin (1 g/day for 3 days) is effective against
multiple drug resistant strains. Shigellosis can be correctly diagnosed in most patients on the
basis of fresh blood in the stool. Neutrophils in fecal smears is also a strongly suggestive sign.
Nonetheless, watery, mucoid diarrhea may be the only symptom of many S sonnei infections,
and any clinical diagnosis should be confirmed by cultivation of the etiologic agent from stools.
Control : Prevention of fecal-oral transmission is the most effective control strategy. Severe
dysentery is treated with ampicillin, trimethoprim-sulfamethoxazole, or, in patients over 17 years
old, a 4-fluorquinolone such as ciprofloxacin. Vaccines are not currently available, but some
promising candidates are being developed.
II.Students practical activities:
1. Prepare smear from pure culture of E.coli, stain with Gram
and microscopy. Estimate the morphology and sketch the
image. Microscopy the smears prepared from Shigella culture
2. Familiarize with diagnostic media for cultivation of E.coli.
Indicate the colonies of lactose-positive and lactose-negative
enterobacteria.
3. Detect the biochemical features of E.coli and Shigella spp.
according to growth results into the Hiss media. Note them in
protocol.
4. Write down the scheme of laboratory diagnostics of the
diarrhea caused by E.coli and bacterial dysentery caused by
Shigella spp...